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The formation of sulfur metabolites during in vitro gastrointestinal digestion of fish, white and red meat is affected by fructo-oligosaccharides

Núria Elias Masiques (UGent) , Els Vossen (UGent) , Stefaan De Smet (UGent) and Thomas Van Hecke (UGent)
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Abstract
Introduction Meat is a source of high quality protein, rich in sulfur-containing amino acids. Various rodent feeding studies indicate that the consumption of heme-Fe or red (vs. white) meat may induce a colonic microbial community shift, accompanied by the formation of sulfur-metabolites (Ijssennagger et al., 2015; Van Hecke et al., 2019, 2021). Hydrogen sulfide (H2S) is a sulfur-metabolite believed to play a key role in intestinal health. Whereas low intestinal H2S levels may be beneficial for intestinal health, high H2S levels may affect the integrity of the mucus layer thus promoting intestinal inflammation (Ijssennagger et al., 2015; Blachier et al., 2021). Different H2S detoxification routes have been proposed: oxidation to sulfate or methylation to methanethiol and dimethyl sulfide (Tangerman 2009). In its turn, methanethiol can be oxidized to dimethyl disulfide and dimethyl trisulfide (Chin & Lindsay, 1994); a reaction which may be catalysed by the pro-oxidant heme-Fe in red meat. The main objective of this study was to assess the formation of sulfur-metabolites produced during in vitro enzymatic gastrointestinal digestion and fermentation of different muscle protein sources (beef, pork, chicken and salmon). These muscle foods were digested with or without fructo-oligosaccharides (FOS), widely found in fruits and vegetables. We hypothesized that the addition of FOS may decrease meat protein fermentation and therefore the formation of sulfur-metabolites. Material & Methods Fresh meats and fish were minced, vacuum packed, cooked in a water bath at 70ºC for 70 min, and frozen stored until digestion in vitro with or without 20% FOS supplemented (in triplicate). The model simulated conditions of the human mouth, stomach, small- and large intestines (Van Hecke et al., 2014, 2018); using four individual fecal donors. Following 24h of fermentation, H2S was determined in the headspace of the digestion vessels by micro GC. Other metabolites (methanethiol, dimethyl x-sulfides, indole, phenol) were determined by using solid phase micro-extraction (SPME) GC-MS. Short chain fatty acids (SCFA) were quantified by GC according to Gadeyne et al. (2016). Ammonia was measured spectrophotometrically (Chaney & Marbach, 1962). Results & Discussion Figure 1 shows the main metabolites produced during in vitro fermentation of the different muscle sources, with or without the addition of FOS. Protein fermentation was significantly reduced when FOS were added to the muscle foods, as indicated by lower levels of ammonia, phenol and indole (all p<0.001). Surprisingly, H2S levels increased with the addition of FOS (p<0.001). The formation of SCFA was higher in the presence of FOS (all p<0.01), hereby acidifying the pH of the ferments (pH 5.6 vs. 6.9). In the colon, lower luminal pH will influence the equilibrium H2S:HS- towards a dominance of the H2S form (Blachier et al., 2017), which might explain the higher H2S levels in the FOS ferments. Yet, H2S concentrations were not significantly different between ferments of salmon, red or white meats. Besides, without FOS added, methanethiol levels were significantly higher in salmon ferments compared to chicken and beef (p<0.001 and p<0.005, respectively). The formation of the different dimethyl x-sulfides was independent of muscle type and was almost completely supressed by the addition of FOS (p<0.001). Conclusion The presence of FOS during fermentation of meat and fish reduced protein fermentation and almost completely prevented the formation of various dimethyl x-sulfides, whereas H2S levels were higher in FOS-added ferments. Salmon ferments contained significantly higher levels of methanethiol compared to chicken and beef ferments absent FOS, whereas other sulfur-metabolites were not affected by the muscle source.

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MLA
Elias Masiques, Núria, et al. “The Formation of Sulfur Metabolites during in Vitro Gastrointestinal Digestion of Fish, White and Red Meat Is Affected by Fructo-Oligosaccharides.” BAMST Symposium 2021, Abstracts, 2021.
APA
Elias Masiques, N., Vossen, E., De Smet, S., & Van Hecke, T. (2021). The formation of sulfur metabolites during in vitro gastrointestinal digestion of fish, white and red meat is affected by fructo-oligosaccharides. BAMST Symposium 2021, Abstracts. Presented at the BAMST Symposium 2021, Online.
Chicago author-date
Elias Masiques, Núria, Els Vossen, Stefaan De Smet, and Thomas Van Hecke. 2021. “The Formation of Sulfur Metabolites during in Vitro Gastrointestinal Digestion of Fish, White and Red Meat Is Affected by Fructo-Oligosaccharides.” In BAMST Symposium 2021, Abstracts.
Chicago author-date (all authors)
Elias Masiques, Núria, Els Vossen, Stefaan De Smet, and Thomas Van Hecke. 2021. “The Formation of Sulfur Metabolites during in Vitro Gastrointestinal Digestion of Fish, White and Red Meat Is Affected by Fructo-Oligosaccharides.” In BAMST Symposium 2021, Abstracts.
Vancouver
1.
Elias Masiques N, Vossen E, De Smet S, Van Hecke T. The formation of sulfur metabolites during in vitro gastrointestinal digestion of fish, white and red meat is affected by fructo-oligosaccharides. In: BAMST Symposium 2021, Abstracts. 2021.
IEEE
[1]
N. Elias Masiques, E. Vossen, S. De Smet, and T. Van Hecke, “The formation of sulfur metabolites during in vitro gastrointestinal digestion of fish, white and red meat is affected by fructo-oligosaccharides,” in BAMST Symposium 2021, Abstracts, Online, 2021.
@inproceedings{01HX991Y7XHHCRRQFDD41MVYD6,
  abstract     = {{Introduction
Meat is a source of high quality protein, rich in sulfur-containing amino acids. Various rodent feeding studies indicate that the consumption of heme-Fe or red (vs. white) meat may induce a colonic microbial community shift, accompanied by the formation of sulfur-metabolites (Ijssennagger et al., 2015; Van Hecke et al., 2019, 2021). Hydrogen sulfide (H2S) is a sulfur-metabolite believed to play a key role in intestinal health. Whereas low intestinal H2S levels may be beneficial for intestinal health, high H2S levels may affect the integrity of the mucus layer thus promoting intestinal inflammation (Ijssennagger et al., 2015; Blachier et al., 2021). Different H2S detoxification routes have been proposed: oxidation to sulfate or methylation to methanethiol and dimethyl sulfide (Tangerman 2009). In its turn, methanethiol can be oxidized to dimethyl disulfide and dimethyl trisulfide (Chin & Lindsay, 1994); a reaction which may be catalysed by the pro-oxidant heme-Fe in red meat. 
The main objective of this study was to assess the formation of sulfur-metabolites produced during in vitro enzymatic gastrointestinal digestion and fermentation of different muscle protein sources (beef, pork, chicken and salmon). These muscle foods were digested with or without fructo-oligosaccharides (FOS), widely found in fruits and vegetables. We hypothesized that the addition of FOS may decrease meat protein fermentation and therefore the formation of sulfur-metabolites.

Material & Methods
Fresh meats and fish were minced, vacuum packed, cooked in a water bath at 70ºC for 70 min, and frozen stored until digestion in vitro with or without 20% FOS supplemented (in triplicate). The model simulated conditions of the human mouth, stomach, small- and large intestines (Van Hecke et al., 2014, 2018); using four individual fecal donors. Following 24h of fermentation, H2S was determined in the headspace of the digestion vessels by micro GC. Other metabolites (methanethiol, dimethyl x-sulfides, indole, phenol) were determined by using solid phase micro-extraction (SPME) GC-MS. Short chain fatty acids (SCFA) were quantified by GC according to Gadeyne et al. (2016). Ammonia was measured spectrophotometrically (Chaney & Marbach, 1962).

Results & Discussion 
Figure 1 shows the main metabolites produced during in vitro fermentation of the different muscle sources, with or without the addition of FOS. Protein fermentation was significantly reduced when FOS were added to the muscle foods, as indicated by lower levels of ammonia, phenol and indole (all p<0.001). Surprisingly, H2S levels increased with the addition of FOS (p<0.001). The formation of SCFA was higher in the presence of FOS (all p<0.01), hereby acidifying the pH of the ferments (pH 5.6 vs. 6.9). In the colon, lower luminal pH will influence the equilibrium H2S:HS- towards a dominance of the H2S form (Blachier et al., 2017), which might explain the higher H2S levels in the FOS ferments. Yet, H2S concentrations were not significantly different between ferments of salmon, red or white meats.
Besides, without FOS added, methanethiol levels were significantly higher in salmon ferments compared to chicken and beef (p<0.001 and p<0.005, respectively). The formation of the different dimethyl x-sulfides was independent of muscle type and was almost completely supressed by the addition of FOS (p<0.001).

Conclusion
The presence of FOS during fermentation of meat and fish reduced protein fermentation and almost completely prevented the formation of various dimethyl x-sulfides, whereas H2S levels were higher in FOS-added ferments. Salmon ferments contained significantly higher levels of methanethiol compared to chicken and beef ferments absent FOS, whereas other sulfur-metabolites were not affected by the muscle source.}},
  author       = {{Elias Masiques, Núria and Vossen, Els and De Smet, Stefaan and Van Hecke, Thomas}},
  booktitle    = {{BAMST Symposium 2021, Abstracts}},
  language     = {{eng}},
  location     = {{Online}},
  title        = {{The formation of sulfur metabolites during in vitro gastrointestinal digestion of fish, white and red meat is affected by fructo-oligosaccharides}},
  year         = {{2021}},
}